發(fā)布時間：2018-06-19 19:28

  本文選題：天然噬菌體抗體庫 + 人源抗體��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2008年博士論文

【摘要】： 上世紀90年代中期以來,抗體藥物在新藥中嶄露頭角。在治療性應用中,全人抗體可以克服鼠源單克隆抗體在臨床應用中的缺點:如誘發(fā)人體產(chǎn)生人抗小鼠抗體(human anti-mouse antibody, HAMA)反應、在人體內半衰期短、不能夠有效地激活CDC及ADCC等。 噬菌體展示抗體庫技術是獲取全人抗體的最主要手段之一。能否從庫中篩選出針對任意抗原的特異性抗體,抗體庫庫容是主要的決定因素。此外,篩選出抗體親和力大小和庫容密切相關。因此,本研究嘗試通過分子生物學技術,經(jīng)Cre/LoxP定位重組系統(tǒng)構建超大容量天然噬菌體抗體庫,并對篩選中得到的抗DR5抗體在原核、真核系統(tǒng)中表達,對表達抗體的抗原結合等特性進行了初步研究。 1人源天然噬菌體抗體庫的建立、篩選 從正常人外周血和新生兒臍血中分離淋巴細胞(180份),提取RNA,用RT-PCR分別擴增抗體可變區(qū)輕重鏈基因(VH和VL),通過重疊PCR技術將VH和VL連接為單鏈抗體ScFv形式,克隆插入到pDF噬菌粒載體,轉化XL1-BLUE細菌得到ScFv初級抗體庫。以高感染復數(shù)(MOI100:1)感染Cre +菌株BS1365,利用Cre/LoxP位點特異性重組原理,使VH和VL基因定向同源重組匹配,隨后以低感染復數(shù)(MOI1:1)感染XL1-BLUE,獲取次級工作庫。結果表明:抗體V區(qū)基因得到有效擴增,初級庫庫容3.6×10~7,工作庫庫容1.8×10~(11),經(jīng)Cre/LoxP定位重組系統(tǒng)成功構建了超大容量天然噬菌體抗體庫;分別用人B淋巴細胞刺激因子(Blys)、人死亡受體胞外段(DR5)、腫瘤壞死因子α(TNFα)、白細胞介素6(IL-6)以及蓖麻毒素(Ricin)等5種不同抗原進行篩選均得到特異性結合的噬菌體抗體,測序結果進一步表明,所獲取抗體基因涵蓋了不同的基因亞群,證明該抗體庫具有良好的多樣性,可用于制備人源抗體。 2在原核、真核系統(tǒng)中表達的抗DR5抗體的抗原結合等特性的初步研究 為了降低試驗費用,縮短實驗周期,借助生物信息學理論及計算機輔助分子模擬技術,通過多序列比對分析、抗體Fv構象穩(wěn)定性分析,對抗體庫中篩選出來的抗DR5單克隆抗體進行了合理性評估。結合篩選過程中的特異性結合實驗結果,以抗體可變區(qū)構象穩(wěn)定性、結合能力、出現(xiàn)頻率為判據(jù)選取C11、B2、A2、A8共四株噬菌體抗體進行生物學實驗。將四株噬菌體抗體以ScFv形式插入pET28a原核表達載體,轉化BL21菌株后表達重組人ScFv抗體。試驗結果表明ScFv抗體分子得到有效表達;此外,超聲破碎后的表達上清,經(jīng)ELISA、Western Blot試驗證實:表達的ScFv抗體蛋白可以和預先純化的DR5抗原特異性結合,證明了抗體庫篩選得到的ScFv抗體具有特異性識別重組人DR5胞外區(qū)蛋白的活性。 將四株噬菌體抗體的輕、重鏈可變區(qū)基因,插入到真核表達載體pCMV-Express中,等量瞬時轉染293T細胞,表達完整的抗體分子。Western blot試驗證實,表達上清中存在能被山羊抗人IgG酶標抗體特異性識別,且分子量與IgG標準品一致的人全分子抗體;并且表達抗體可特異性識別轉印在NC膜上的DR5抗原;競爭ELISA結果顯示,表達抗體與DR5的天然配體TRAIL存在競爭關系,其結合力隨TRAIL劑量增加而下降。
[Abstract]:In the mid 90s of the last century, antibody drugs have emerged in new drugs. In therapeutic applications, human antibodies can overcome the shortcomings of mouse monoclonal antibodies in clinical application: such as inducing human human anti mouse antibody (human anti-mouse antibody, HAMA) reaction, short half life in human body, and not effectively activating CDC and ADC C and so on.
Phage display antibody library technology is one of the most important means of obtaining all human antibodies. Whether the specific antibody against any antigen is screened from the library, the capacity of the antibody library is the main determinant. In addition, the size of the antibody affinity is closely related to the size of the antibody. Therefore, this study tries to pass the molecular biology technology through the molecular biology technology, Cre/LoxP A large capacity natural phage antibody library was constructed by positioning and recombination system, and the characteristics of anti DR5 antibody in the prokaryotic, eukaryotic system and antigen binding of the expressed antibody were preliminarily studied.
Establishment of a natural phage antibody library of 1 human sources and screening
Lymphocytes (180 portions) were isolated from normal human peripheral blood and neonatal umbilical blood, and RNA was extracted and amplified by RT-PCR (VH and VL). VH and VL were linked to ScFv form of single chain antibody by overlapping PCR technology. The clones were inserted into the pDF phagocytic carrier and XL1-BLUE bacteria were converted to ScFv primary antibody library. The number (MOI100:1) infected with Cre + strain BS1365, using the principle of Cre/LoxP specific recombination, matching the VH and VL genes to homologous recombination, and then using the low infection complex (MOI1:1) to infect XL1-BLUE to obtain the secondary working library. The results showed that the antibody V region gene was effectively amplified, the storage capacity of the primary reservoir was 3.6 * 10~7, the storage capacity of the working library was 1.8 x 10~ (11), and Cre/Lo The xP location and recombination system successfully constructed a large capacity natural phage antibody library, and screened 5 different antigens, such as human B lymphocyte stimulator (Blys), human death receptor extracellular segment (DR5), tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6) and ricin (Ricin). The sequence results further showed that the antibody genes covered different subsets of genes, which showed that the antibody library had good diversity and could be used for the preparation of human antibody.
2 preliminary study on antigen binding characteristics of anti DR5 antibodies expressed in prokaryotic and eukaryotic systems
In order to reduce the test cost and shorten the experimental period, by means of bioinformatics theory and computer aided molecular simulation technology, the Conformation Stability Analysis of antibody Fv was analyzed by multi sequence alignment, and the rationality evaluation of anti DR5 monoclonal antibodies screened from the antagonism body library was conducted. Four phage antibodies of C11, B2, A2 and A8 were selected for biological experiments. The four phage antibodies were inserted into the pET28a prokaryotic expression vector in the form of ScFv, and the BL21 strain was transformed to express the recombinant human ScFv antibody. The experimental results showed that the ScFv antibody molecules were expressed effectively. In addition, the expression supernatant after ultrasonic fragmentation was confirmed by ELISA, Western Blot test. The expression of ScFv antibody protein can be specifically combined with the pre purified DR5 antigen. It is proved that the ScFv antibody screened by the antibody library has the specific activity to identify the recombinant human DR5 extracellular domain protein.
The light, heavy chain variable region gene of four phage antibodies was inserted into the eukaryotic expression vector pCMV-Express, and the 293T cells were transiently transfected in the same amount. The expression of the complete antibody molecule.Western blot test proved that the expression of the expression supernatant could be identified by the Goat anti human IgG specific antibody specific recognition, and the molecular weight was consistent with the IgG standard. The antibody can be expressed specifically to identify the DR5 antigen transferred to the NC membrane; competitive ELISA results show that the expression antibody has a competitive relationship with the natural ligand TRAIL of DR5, and its binding force decreases with the increase of TRAIL dose.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392

【參考文獻】

相關期刊論文 前6條

1 白慧玲,龔非力,馬遠方;死亡受體5誘導Jurkat細胞凋亡的作用[J];第四軍醫(yī)大學學報;2005年22期

2 白慧玲;杜耀武;王雪垠;李淑蓮;王靖;劉廣超;馬遠方;;交聯(lián)抗DR5單抗YM366EC誘導的Jurkat細胞凋亡機制探討[J];現(xiàn)代免疫學;2007年04期

3 ;A New Combination of Mutated loxPs in a Vector for Construction of Phage Antibody Libraries[J];Acta Biochimica et Biophysica Sinica;2005年07期

4 唐曉明,王清明,楊俊濤,陳吉中,范國才,汪思應;大容量人天然抗體庫的構建、鑒定及初步應用[J];中國生物工程雜志;2005年10期

5 馬遠方,楊東亮,陳有海;Analysis of TRAIL receptor expression using anti-TRAIL death receptor-5 monoclonal antibodies[J];Chinese Medical Journal;2003年06期

6 王琰,化冰,劉群英,高榮凱,朱迎春,陳宇萍;隨機化CDR3抗體庫的構建及不經(jīng)免疫制備抗體的初步探索[J];中華微生物學和免疫學雜志;1997年06期

，

本文編號：2041012